Editing 5-Methylcytosine

{{short description|Chemical compound which is a modified DNA base}}
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| InChIKey = LRSASMSXMSNRBT-UHFFFAOYAO
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| SMILES = O=C1/N=C\C(=C(\N)N1)C
| MeSHName=5-Methylcytosine
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'''5-Methylcytosine''' ('''5mC''') is a [[methylation|methylated]] form of the [[DNA]] base [[cytosine]] (C) that regulates gene [[Transcription (genetics)|transcription]] and takes several other biological roles.<ref name=":3">{{cite journal|last1=Wu|first1=Xiaoji|last2=Zhang|first2=Yi|date=2017-05-30|title=TET-mediated active DNA demethylation: mechanism, function and beyond|journal=Nature Reviews Genetics|volume=18|issue=9|pages=517–534|doi=10.1038/nrg.2017.33|pmid=28555658|s2cid=3393814|issn=1471-0056}}</ref> When cytosine is methylated, the DNA maintains the same sequence, but the [[Gene expression#DNA methylation and demethylation in transcriptional regulation|expression of methylated genes]] can be altered (the study of this is part of the field of [[epigenetics]]). 5-Methylcytosine is incorporated in the [[nucleoside]] [[5-Methylcytidine|5-methylcytidine]].

== Discovery ==
While trying to isolate the bacterial [[toxin]] responsible for [[tuberculosis]], W.G. Ruppel isolated a novel [[nucleic acid]] named [[tuberculinic acid]] in 1898 from ''[[Mycobacterium tuberculosis|Tubercle bacillus]]''.<ref>{{cite book |vauthors = Matthews AP |year=2012 |url=https://books.google.com/books?id=809EPAAACAAJ|title= Physiological Chemistry |publisher= Williams & Wilkins Company/ |pages=167 |isbn=978-1130145373}}</ref> The nucleic acid was found to be unusual, in that it contained in addition to [[thymine]], [[guanine]] and [[cytosine]], a methylated nucleotide. In 1925, [[Treat Baldwin Johnson|Johnson]] and Coghill successfully detected a minor amount of a methylated cytosine derivative as a product of [[hydrolysis]] of tuberculinic acid with [[sulfuric acid]].<ref>{{cite journal |vauthors = Johnson TB, Coghill RD |year=1925 |title= The discovery of 5-methyl-cytosine in tuberculinic acid, the nucleic acid of the ''Tubercle bacillus'' |journal=  J Am Chem Soc|volume=47 |issue=11|pages=2838–2844|doi=10.1021/ja01688a030}}</ref><ref>Grosjean H (2009). [https://www.ncbi.nlm.nih.gov/books/NBK6489/#A79016 Nucleic Acids Are Not Boring Long Polymers of Only Four Types of Nucleotides: A Guided Tour]. Landes Bioscience.</ref> This report was severely criticized because their identification was based solely on the optical properties of the crystalline [[picrate]], and other scientists failed to reproduce the same result.<ref>{{cite journal |vauthors = Vischer E, Zamenhof S, Chargaff E |year=1949 |title= Microbial nucleic acids: the desoxypentose nucleic acids of avian tubercle bacilli and yeast|journal= J Biol Chem|volume=177 |issue=1|pages=429–438|doi=10.1016/S0021-9258(18)57100-3 |pmid=18107446|doi-access=free}}</ref>  But its existence was ultimately proven in 1948, when [[Rollin Douglas Hotchkiss|Hotchkiss]] separated the nucleic acids of [[DNA]] from [[calf (animal)|calf]] [[thymus]] using [[paper chromatography]], by which he detected a unique methylated cytosine, quite distinct from conventional cytosine and [[uracil]].<ref>{{cite journal |vauthors = Hotchkiss RD |year=1948|title= The quantitative separation of purines, pyrimidines and nucleosides by paper chromatography|journal=J Biol Chem|volume=175 |issue=1|pages=315–332|doi=10.1016/S0021-9258(18)57261-6|pmid=18873306|doi-access=free}}</ref> After seven decades, it turned out that it is also a common feature in different [[RNA]] molecules, although the precise role is uncertain.<ref>{{cite journal |vauthors = Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T|year=2012|title= Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA|journal=Nucleic Acids Res|volume=40 |issue=11|pages=5023–5033|pmid=22344696|doi=10.1093/nar/gks144|pmc=3367185}}</ref>

== ''In vivo'' ==
The function of this chemical varies significantly among species:<ref>{{cite journal |vauthors =Colot V, Rossignol JL |title=Eukaryotic DNA methylation as an evolutionary device |journal=BioEssays |volume=21 |issue=5 |pages=402–411 |year=1999 |pmid=10376011 |doi=10.1002/(SICI)1521-1878(199905)21:5<402::AID-BIES7>3.0.CO;2-B|s2cid=10784130 }}</ref>
* In bacteria, 5-methylcytosine can be found at a variety of sites, and is often used as a marker to protect DNA from being cut by native methylation-sensitive [[restriction enzymes]].
* In plants, 5-methylcytosine occurs at [[CpG site|CpG]], CpHpG and CpHpH sequences (where H = A, C or T).
* In fungi and animals, 5-methylcytosine predominantly occurs at [[CpG site|CpG]] dinucleotides. Most [[eukaryote]]s methylate only a small percentage of these sites, but 70-80% of CpG cytosines are methylated in [[vertebrate]]s. In mammalian cells, clusters of CpG at the 5' ends of genes are termed CpG islands.<ref>{{cite journal|last=Bird|first=Adrian P.|date=May 1986|title=CpG-rich islands and the function of DNA methylation|journal=Nature|volume=321|issue=6067|pages=209–213|doi=10.1038/321209a0|pmid=2423876|issn=0028-0836|bibcode=1986Natur.321..209B|s2cid=4236677}}</ref> 1% of all mammalian DNA is 5mC.<ref>{{cite journal|last1=Ehrlich|first1=M.|last2=Wang|first2=R. Y.|date=1981-06-19|title=5-Methylcytosine in eukaryotic DNA|journal=Science|language=en|volume=212|issue=4501|pages=1350–1357|doi=10.1126/science.6262918|issn=0036-8075|pmid=6262918|bibcode=1981Sci...212.1350E}}</ref>

While spontaneous [[deamination]] of cytosine forms [[uracil]], which is recognized and removed by DNA repair enzymes, deamination of 5-methylcytosine forms [[thymine]]. This conversion of a DNA base from cytosine (C) to thymine (T) can result in a [[Transition (genetics)|transition mutation]].<ref>{{cite journal |vauthors =Sassa A, Kanemaru Y, Kamoshita N, Honma M, Yasui M |title=Mutagenic consequences of cytosine alterations site-specifically embedded in the human genome |journal=Genes and Environment |volume=38 |issue=1 |pages=17 |year=2016 |pmid=27588157 |doi=10.1186/s41021-016-0045-9 |pmc=5007816 |doi-access=free |bibcode=2016GeneE..38...17S }}</ref> In addition, active enzymatic deamination of cytosine or 5-methylcytosine by the [[APOBEC]] family of cytosine deaminases could have beneficial implications on various cellular processes as well as on organismal evolution.<ref name="Chahwan R, Wontakal SN, and Roa S">{{cite journal| vauthors= Chahwan R, Wontakal SN, Roa S| title=Crosstalk between genetic and epigenetic information through cytosine deamination| journal=Trends in Genetics| volume = 26| pages = 443–448| year = 2010 | doi = 10.1016/j.tig.2010.07.005| pmid = 20800313| issue = 10}}</ref> The implications of deamination on [[5-hydroxymethylcytosine]], on the other hand, remains less understood.

== ''In vitro'' ==
The NH<sub>2</sub> group can be removed (deamination) from 5-methylcytosine to form [[thymine]] with use of reagents such as [[nitrous acid]]; cytosine deaminates to uracil (U) under similar conditions.{{cn|date=March 2024}}

[[File:Deamination 5-Methylcytosine to Thymine.svg|300px|thumb|none|Deamination of 5-methylcytosine to thymine]]

5-methylcytosine is resistant to deamination by [[bisulfite]] treatment, which deaminates cytosine residues. This property is often exploited to analyze DNA cytosine methylation patterns with [[bisulfite sequencing]].<ref>{{cite journal |vauthors =Clark SJ, Harrison J, Paul CL, Frommer M |title=High sensitivity mapping of methylated cytosines |journal=Nucleic Acids Res. |volume=22 |issue=15 |pages=2990–2997 |year=1994 |pmid=8065911 |doi=10.1093/nar/22.15.2990 |pmc=310266}}</ref>

== Addition and regulation with DNMTs (Eukaryotes) ==
5mC marks are placed on genomic DNA via [[DNA methyltransferase]]s (DNMTs). There are 5 DNMTs in humans: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L, and in algae and fungi 3 more are present (DNMT4, DNMT5, and DNMT6).<ref>{{cite journal|last1=Ponger|first1=Loïc|last2=Li|first2=Wen-Hsiung|date=2005-04-01|title=Evolutionary Diversification of DNA Methyltransferases in Eukaryotic Genomes|url=https://academic.oup.com/mbe/article/22/4/1119/1083517|journal=Molecular Biology and Evolution|language=en|volume=22|issue=4|pages=1119–1128|doi=10.1093/molbev/msi098|pmid=15689527|issn=0737-4038|doi-access=free}}</ref> DNMT1 contains the replication foci targeting sequence (RFTS) and the CXXC domain which catalyze the addition of 5mC marks. RFTS directs DNMT1 to loci of DNA replication to assist in the maintenance of 5mC on daughter strands during DNA replication, whereas CXXC contains a [[zinc finger]] domain for ''de novo'' addition of methylation to the DNA.<ref name=":0">{{cite journal|last=Lyko|first=Frank|date=February 2018|title=The DNA methyltransferase family: a versatile toolkit for epigenetic regulation|journal=Nature Reviews Genetics|language=en|volume=19|issue=2|pages=81–92|doi=10.1038/nrg.2017.80|pmid=29033456|s2cid=23370418|issn=1471-0064}}</ref> DNMT1 was found to be the predominant DNA methyltransferase in all human tissue.<ref name=":1">{{cite journal|last1=Robertson|first1=K D|last2=Uzvolgyi|first2=E|last3=Liang|first3=G|last4=Talmadge|first4=C|last5=Sumegi|first5=J|last6=Gonzales|first6=F A|last7=Jones|first7=P A|date=1999-06-01|title=The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors.|journal=Nucleic Acids Research|volume=27|issue=11|pages=2291–2298|issn=0305-1048|pmid=10325416|pmc=148793|doi=10.1093/nar/27.11.2291}}</ref> Primarily, DNMT3A and DNMT3B are responsible for ''de novo'' methylation, and DNMT1 maintains the 5mC mark after replication.<ref name=":3" /> DNMTs can interact with each other to increase methylating capability. For example, 2 DNMT3L can form a complex with 2 DNMT3A to improve interactions with the DNA, facilitating the methylation.<ref>{{cite journal|last1=Jia|first1=Da|last2=Jurkowska|first2=Renata Z.|last3=Zhang|first3=Xing|last4=Jeltsch|first4=Albert|last5=Cheng|first5=Xiaodong|date=September 2007|title=Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation|journal=Nature|language=en|volume=449|issue=7159|pages=248–251|doi=10.1038/nature06146|pmid=17713477|pmc=2712830|issn=1476-4687|bibcode=2007Natur.449..248J}}</ref> Changes in the expression of DNMT results in aberrant methylation. Overexpression produces increased methylation, whereas disruption of the enzyme decreased levels of methylation.<ref name=":1" />
[[File:DNMT reaction mechanism.tif|alt=DNMT reaction mechanism|left|thumb|595x595px|Addition of methyl group to cytosine]]

The mechanism of the addition is as follows: first a cysteine residue on the DNMT's PCQ motif creates a nucleophillic attack at carbon 6 on the cytosine nucleotide that is to be methylated. [[S-Adenosyl methionine|S-Adenosylmethionine]] then donates a methyl group to carbon 5. A base in the DNMT enzyme deprotonates the residual hydrogen on carbon 5 restoring the double bond between carbon 5 and 6 in the ring, producing the 5-methylcytosine base pair.<ref name=":0" />

== Demethylation ==
After a cytosine is methylated to 5mC, it can be reversed back to its initial state via multiple mechanisms. Passive DNA demethylation by dilution eliminates the mark gradually through replication by a lack of maintenance by DNMT. In active DNA demethylation, a series of oxidations converts it to [[5-hydroxymethylcytosine]] (5hmC), [[5-Formylcytosine|5-formylcytosine]] (5fC), and 5-carboxylcytosine (5caC), and the latter two are eventually excised by [[Thymine-DNA glycosylase|thymine DNA glycosylase]] (TDG), followed by base excision repair (BER) to restore the cytosine.<ref name=":3" /> TDG knockout produced a 2-fold increase of 5fC without any statistically significant change to levels of 5hmC, indicating 5mC must be iteratively oxidized at least twice before its full demethylation.<ref>{{cite journal|last1=Song|first1=Chun-Xiao|last2=Szulwach|first2=Keith E.|last3=Dai|first3=Qing|last4=Fu|first4=Ye|last5=Mao|first5=Shi-Qing|last6=Lin|first6=Li|last7=Street|first7=Craig|last8=Li|first8=Yujing|last9=Poidevin|first9=Mickael|last10=Wu|first10=Hao|last11=Gao|first11=Juan|date=2013-04-25|title=Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming|journal=Cell|volume=153|issue=3|pages=678–691|doi=10.1016/j.cell.2013.04.001|issn=1097-4172|pmc=3657391|pmid=23602153}}</ref> The oxidation occurs through the [[TET enzymes|TET]] (Ten-eleven translocation) family dioxygenases ([[TET enzymes]]) which can convert 5mC, 5hmC, and 5fC to their oxidized forms. However, the enzyme has the greatest preference for 5mC and the initial reaction rate for 5hmC and 5fC conversions with TET2 are 4.9-7.6 fold slower.<ref>{{cite journal|last1=Ito|first1=Shinsuke|last2=Shen|first2=Li|last3=Dai|first3=Qing|last4=Wu|first4=Susan C.|last5=Collins|first5=Leonard B.|last6=Swenberg|first6=James A.|last7=He|first7=Chuan|last8=Zhang|first8=Yi|date=2011-09-02|title=Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine|journal=Science|language=en|volume=333|issue=6047|pages=1300–1303|doi=10.1126/science.1210597|issn=0036-8075|pmid=21778364|pmc=3495246|bibcode=2011Sci...333.1300I}}</ref> TET requires Fe(II) as cofactor, and oxygen and [[α-ketoglutarate]] (α-KG) as substrates, and the latter substrate is generated from isocitrate by the enzyme [[isocitrate dehydrogenase]] (IDH).<ref>{{cite journal|last1=Lu|first1=Xingyu|last2=Zhao|first2=Boxuan Simen|last3=He|first3=Chuan|date=2015-02-12|title=TET Family Proteins: Oxidation Activity, Interacting Molecules, and Functions in Diseases|journal=Chemical Reviews|volume=115|issue=6|pages=2225–2239|doi=10.1021/cr500470n|pmid=25675246|pmc=4784441|issn=0009-2665}}</ref> Cancer however can produce [[2-hydroxyglutarate]] (2HG) which competes with α-KG, reducing TET activity, and in turn reducing conversion of 5mC to 5hmC.<ref>{{cite journal|last1=Xu|first1=Wei|last2=Yang|first2=Hui|last3=Liu|first3=Ying|last4=Yang|first4=Ying|last5=Wang|first5=Ping|last6=Kim|first6=Se-Hee|last7=Ito|first7=Shinsuke|last8=Yang|first8=Chen|last9=Wang|first9=Pu|last10=Xiao|first10=Meng-Tao|last11=Liu|first11=Li-xia|date=2011-01-18|title=Oncometabolite 2-Hydroxyglutarate Is a Competitive Inhibitor of α-Ketoglutarate-Dependent Dioxygenases|journal=Cancer Cell|volume=19|issue=1|pages=17–30|doi=10.1016/j.ccr.2010.12.014|issn=1535-6108|pmc=3229304|pmid=21251613}}</ref>

== Role in humans ==
=== In cancer ===
In cancer, DNA can become both overly methylated, termed [[hypermethylation]], and under-methylated, termed hypomethylation.<ref name=":22">{{cite journal|last=Ehrlich|first=Melanie|date=2009-12-01|title=DNA hypomethylation in cancer cells|journal=Epigenomics|volume=1|issue=2|pages=239–259|doi=10.2217/epi.09.33|issn=1750-1911|pmc=2873040|pmid=20495664}}</ref> CpG islands overlapping gene promoters are ''de novo'' methylated resulting in aberrant inactivation of genes normally associated with growth inhibition of tumors (an example of hypermethylation).<ref>{{cite journal|last=Jones|first=Peter A.|date=1996-06-01|title=DNA Methylation Errors and Cancer|url=https://cancerres.aacrjournals.org/content/56/11/2463|journal=Cancer Research|language=en|volume=56|issue=11|pages=2463–2467|issn=0008-5472|pmid=8653676}}</ref> Comparing tumor and normal tissue, the former had elevated levels of the methyltransferases DNMT1, DNMT3A, and mostly DNMT3B, all of which are associated with the abnormal levels of 5mC in cancer.<ref name=":1" /> Repeat sequences in the genome, including satellite DNA, Alu, and long interspersed elements (LINE), are often seen hypomethylated in cancer, resulting in expression of these normally silenced genes, and levels are often significant markers of tumor progression.<ref name=":22" /> It has been hypothesized that there a connection between the hypermethylation and hypomethylation; over activity of DNA methyltransferases that produce the abnormal ''de novo'' 5mC methylation may be compensated by the removal of methylation, a type of epigenetic repair. However, the removal of methylation is inefficient resulting in an overshoot of genome-wide hypomethylation. The contrary may also be possible; over expression of hypomethylation may be silenced by genome-wide hypermethylation.<ref name=":22" />  Cancer hallmark capabilities are likely acquired through epigenetic changes that alter the 5mC in both the cancer cells and in surrounding tumor-associated stroma within the tumor microenvironment.<ref>{{cite journal|last1=Hanahan|first1=Douglas|last2=Weinberg|first2=Robert A.|date=2011-03-04|title=Hallmarks of Cancer: The Next Generation|journal=Cell|language=en|volume=144|issue=5|pages=646–674|doi=10.1016/j.cell.2011.02.013|issn=0092-8674|pmid=21376230|doi-access=free|url=https://infoscience.epfl.ch/record/171696/files/1-s2.0-S0092867411001279-main.pdf}}</ref> The anticancer drug [[Cisplatin]] has been reported to react with 5mC.<ref>{{cite journal|last1=Menke|first1=Annika|last2=Dubini|first2=Romeo C.A.|last3=Mayer|first3=Peter|last4=Rovó|first4=Petra|last5=Daumann|first5=Lena|date=2020-10-23|title=Formation of Cisplatin Adducts with the Epigenetically-relevant Nucleobase 5-Methylcytosine|journal=European Journal of Inorganic Chemistry|volume=2021|pages=30–36|doi=10.1002/ejic.202000898|issn=1434-1948|doi-access=free}}</ref>

=== As a biomarker of aging ===
"Epigenetic age" refers to the connection between chronological age and levels of DNA methylation in the genome.<ref>{{cite journal|last1=Horvath|first1=Steve|last2=Raj|first2=Kenneth|date=June 2018|title=DNA methylation-based biomarkers and the epigenetic clock theory of ageing|journal=Nature Reviews Genetics|language=en|volume=19|issue=6|pages=371–384|doi=10.1038/s41576-018-0004-3|pmid=29643443|s2cid=4709691|issn=1471-0064}}</ref> Coupling the levels of DNA methylation, in specific sets of CpGs called "clock CpGs", with algorithms that regress the typical levels of collective genome-wide methylation at a given chronological age, allow for epigenetic age prediction. During youth (0–20 years old), changes in DNA methylation occur at a faster rate as development and growth progresses, and the changes begin to slow down at older ages. Multiple epigenetic age estimators exist. Horvath's clock measures a multi-tissue set of 353 CpGs, half of which positively correlate with age, and the other half negatively, to estimate the epigenetic age.<ref>{{cite journal|last=Horvath|first=Steve|date=2013-12-10|title=DNA methylation age of human tissues and cell types|journal=Genome Biology|volume=14|issue=10|pages=3156|doi=10.1186/gb-2013-14-10-r115|issn=1474-760X|pmc=4015143|pmid=24138928 |doi-access=free }}{{Erratum|doi=10.1186/s13059-015-0649-6|pmid=25968125|http://retractionwatch.com/2015/06/15/high-profile-aging-paper-posts-old-erratum-requested-by-author-more-than-one-year-prior/ ''Retraction Watch''|checked=yes}}</ref> Hannum's clock utilizes adult blood samples to calculate age based on an orthogonal basis of 71 CpGs.<ref>{{cite journal|last1=Hannum|first1=Gregory|last2=Guinney|first2=Justin|last3=Zhao|first3=Ling|last4=Zhang|first4=Li|last5=Hughes|first5=Guy|last6=Sadda|first6=SriniVas|last7=Klotzle|first7=Brandy|last8=Bibikova|first8=Marina|last9=Fan|first9=Jian-Bing|last10=Gao|first10=Yuan|last11=Deconde|first11=Rob|date=2013-01-24|title=Genome-wide Methylation Profiles Reveal Quantitative Views of Human Aging Rates|journal=Molecular Cell|volume=49|issue=2|pages=359–367|doi=10.1016/j.molcel.2012.10.016|issn=1097-2765|pmc=3780611|pmid=23177740}}</ref> Levine's clock, known as DNAm PhenoAge, depends on 513 CpGs and surpasses the other age estimators in predicting mortality and lifespan, yet displays bias with non-blood tissues.<ref>{{cite journal|last1=Levine|first1=Morgan E.|last2=Lu|first2=Ake T.|last3=Quach|first3=Austin|last4=Chen|first4=Brian H.|last5=Assimes|first5=Themistocles L.|last6=Bandinelli|first6=Stefania|last7=Hou|first7=Lifang|last8=Baccarelli|first8=Andrea A.|last9=Stewart|first9=James D.|last10=Li|first10=Yun|last11=Whitsel|first11=Eric A.|date=2018-04-17|title=An epigenetic biomarker of aging for lifespan and healthspan|journal=Aging (Albany NY)|volume=10|issue=4|pages=573–591|doi=10.18632/aging.101414|issn=1945-4589|pmc=5940111|pmid=29676998}}</ref> There are reports of age estimators with the methylation state of only one CpG in the gene ELOVL2.<ref>{{cite journal|last1=Garagnani|first1=Paolo|last2=Bacalini|first2=Maria G.|last3=Pirazzini|first3=Chiara|last4=Gori|first4=Davide|last5=Giuliani|first5=Cristina|last6=Mari|first6=Daniela|last7=Blasio|first7=Anna M. Di|last8=Gentilini|first8=Davide|last9=Vitale|first9=Giovanni|last10=Collino|first10=Sebastiano|last11=Rezzi|first11=Serge|date=2012|title=Methylation of ELOVL2 gene as a new epigenetic marker of age|journal=Aging Cell|language=en|volume=11|issue=6|pages=1132–1134|doi=10.1111/acel.12005|pmid=23061750|issn=1474-9726|hdl=11585/128353|s2cid=8775590|hdl-access=free}}</ref> Estimation of age allows for prediction lifespan through expectations of age related conditions that individuals may be subject to based on their 5mC methylation markers.{{cn|date=March 2024}}

== References ==
{{reflist}}

== Literature ==
* {{cite book |author =Griffiths, Anthony J. F. |title=An Introduction to genetic analysis |publisher=W.H. Freeman |location=San Francisco |year=1999 |pages=Chapter 15: Gene Mutation |isbn=0-7167-3520-2 |no-pp=true}} ([https://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=iga.TOC available online] at the United States [[National Center for Biotechnology Information]])

{{DEFAULTSORT:Methylcytosine5}}
[[Category:Nucleobases]]
[[Category:Pyrimidones]]
[[Category:Biomarkers]]
[[Category:Methyl compounds]]